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Tag and method for simultaneously visualizing DNAs, mRNAs and proteins of genes in living cells

A living cell and gene technology, applied in the field of mRNA and protein labeling, DNA, can solve the problems of lack of, can not clearly capture the quantitative analysis of nascent RNA, lack of regulation of endogenous gene expression, etc.

Active Publication Date: 2020-09-29
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

This work visualizes both mature mRNA and nascent RNA, thus failing to clearly capture nascent RNA production and quantify the dynamic regulation of transcription
In addition, the DNA of the three components of the reporter system is ~20kb in total, which can only be used as an exogenous reporter gene, which limits the application of this technology to a certain extent
[0005] Therefore, the existing technology lacks a live cell imaging technology that can be widely used to realize the visualization of DNA, mRNA and protein of genes at the same time, and lacks the ability to better study the expression regulation of endogenous genes. and other research work provide a powerful gene visualization tool

Method used

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  • Tag and method for simultaneously visualizing DNAs, mRNAs and proteins of genes in living cells
  • Tag and method for simultaneously visualizing DNAs, mRNAs and proteins of genes in living cells
  • Tag and method for simultaneously visualizing DNAs, mRNAs and proteins of genes in living cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] S1. Design and construct a blue fluorescent protein sequence containing introns:

[0068] The human HeLa cell genome was extracted, and the blue fluorescent protein sequence containing introns was obtained by nucleic acid synthesis, such as figure 1 as shown in (a);

[0069] S2. The screened TS1 capable of efficiently targeting and editing the nematode genome 12x Sequence with reduced repetitiveness of MS2V5 12x The sequence is synthesized by nucleic acid, and the TS1 repeat unit and the MS2V5 repeat unit are alternately connected to form a DNA / RNA dual visualization sequence, such as figure 1 as shown in (b);

[0070] S3. Construction of TriTag tags: Insert the dual visualization sequence into the intron region of the blue fluorescent protein sequence containing introns to obtain TriTag tags using the enzyme-cut ligation method, such as figure 1 As shown in (c), specifically the BstxI restriction enzyme site inserted into the intron sequence.

Embodiment 2

[0072] (1) Construct a visualized stable cell line, such as figure 2 As shown in (a):

[0073] (1.1) On the same day, HEK293T cells were cultured with PS-free medium and spread into a 12-well plate to form the first cell culture medium. The concentration of HEK293T cells added could make the cell density reach more than 80% the next day;

[0074] (1.2) The next day, carry out lentiviral packaging: use 75 μl of serum-reduced medium (Opti-medium) to premix each 750ng of dCas9-GFP 14xAfter virus expression plasmid and stdMCP-tdTomato virus expression plasmid, pCMV-dR8.91 plasmid of 705ng and PMD2.G plasmid of 87ng, then add 4.5μl Fugene transient transfection reagent (Promega) to form liposome, add dropwise to the In the supernatant of the first cell culture medium, HEK293T cells in the first cell culture medium were transiently transfected to prepare cells containing virus particles;

[0075] (1.3) After 12 hours of transient transfection in step (1.2), suck off the supernata...

Embodiment 3

[0100] (1) Construct the target endogenous gene (human histone H2B gene and nuclear membrane protein LMNA gene) visualization system in a stable cell line and transfect and sort:

[0101] (1.1) According to the protein structure prediction of the target endogenous gene product H2B histone and LMNA nuclear membrane protein, choose to insert the TriTag tag into the C-terminus of the human histone H2B gene, and insert the TriTag tag into the N of the human nuclear membrane protein LMNA gene end. The human HeLa cell genome was extracted, and the homology arm sequences (5' homology arm sequence and 3' homology arm sequence) on both sides of the H2B gene C-terminus or LMNA gene N-terminus were respectively amplified, and the length of the homology arm sequence was about 500 bp. The HDR repair template of 5' homology arm sequence-TriTag tag-3' homology arm sequence was constructed by homologous recombination. like image 3 (a) shown.

[0102] (1.2) As in steps (2.2)-(2.4) in Examp...

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Abstract

The invention discloses a triple tag and method for simultaneously visualizing DNAs, mRNAs and proteins of genes in living cells. The tag comprises a CRISPR-Tag DNA sequence composed of 12 TS1 repeating units, an MS2 DNA sequence composed of 12 MS2V5 unit sequences, and a blue fluorescent protein sequence containing an intron. The DNA sequences are short, and can be inserted into an N terminal ora C terminal of a target protein coding gene in a CRISPR-Cas9-mediated gene recombination manner. A preparation method comprises the steps of firstly, preparing a TriTag tag; secondly, constructing avisualized stable cell line; thirdly, constructing a target endogenous gene visualization system and carrying out transfection and sorting; and finally achieving dynamic imaging of the living cells. The TriTag tag disclosed by the invention can be tagged, achieves dynamic imaging of chromatins, can visually visualize chromatin dynamics and gene expression of endogenous specific target genes, is conducive to data integration and analysis and helps analyzing a dynamic regulation mechanism of the gene expression.

Description

technical field [0001] The invention relates to a method for establishing a TriTag labeling imaging system in the field of biotechnology, and its specific content relates to a label and method for simultaneously visualizing DNA, mRNA and protein of genes in living cells, so as to realize real-time imaging and analysis of gene expression regulation. Background technique [0002] Gene expression is the process of transferring genetic information from gene to protein. As two key steps of gene expression, transcription and translation are closely regulated throughout the process. However, few studies have visualized the entire process of gene expression regulation: that is, in living cells, the DNA, mRNA and protein of genes are visualized at the same time, revealing how genes regulate transcription at the chromatin level, and then realize the spatiotemporal regulation of protein expression. [0003] There has been a lot of work to visualize RNA in living cells. In these works,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/113C12N15/90C12N15/867C12N15/66C12N15/65C12N9/22C12N5/10
CPCC12N9/22C12N15/113C12N15/65C12N15/66C12N15/86C12N15/907C12N2740/15043C12N2310/20
Inventor 陈宝惠徐海月邹炜
Owner ZHEJIANG UNIV
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